Microscope sample preparation device

ABSTRACT

A sample preparation device for electron microscopy (EM) that is configured to eliminate user-to-user variations and environment contaminations, which are often present in the conventional method of sample preparation. The device not only provides a means for evenly and reproducibly delivering a fluid or sample to an EM grid, but also provides a means for sealing the EM grid in an air-tight chamber and delivering air-sensitive samples to the EM grid. The platform may comprise readily fabricated glass chips with features integrated to preserve the integrity of the sample grid and to facilitate its extraction. The methods may eliminate the element of user dependent variability and thus improve the throughput, reproducibility and translation of these methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional application is a continuation of and claimspriority to non-provisional application Ser. No. 15/263,949, entitled“MICROSCOPE SAMPLE PREPARATION DEVICE,” filed Sep. 13, 2016 by the sameinventors, which claims priority to provisional application No.62/236,368, entitled “ELECTRON MICROSCOPE SAMPLE PREPARATION DEVICE,”filed Oct. 2, 2015 by the same inventors.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates, generally, to the field of microscopy. Morespecifically, it relates to preparation of samples for electronmicroscopy.

2. Brief Description of the Prior Art

Preparation of high quality samples is critical for structuredetermination of biomolecules. Sample preparation for negative stain EMis typically done by hand and consists of a series of blotting steps ofboth the sample and heavy metal stain. The stain is used to introducecontrast to the images and to lock the native structure of the proteininto place.

Conventional negative staining of samples on EM grids is the primarymethod used by most EM labs to evaluate their samples and can be theonly method for specimen preparation of small or highly heterogeneoussamples. There are almost as many protocols for making negative staingrids as there are EM labs, and researchers adhere to their ownprotocols that have been developed for years. For example, one protocol,as depicted in FIG. 1, might call for retrieving EM grid 102 usingtweezers 104, hand pipetting microliter sample 103 onto thecarbon-coated copper grid 102, hand blotting sample 103 with absorbentfilter paper 106 to remove most of the liquid, hand pipetting amicroliter volume of heavy metal stain 105 onto EM grid 102, and againhand blotting EM grid 102 with absorbent paper 106 to remove excessstain. Alternately, other labs advocate transferring a grid betweendifferent drops of sample, water, and stain and then blotting.

There are several disadvantages to the conventional method of preparingan EM sample by hand. First, the grid and sample are open to theenvironment, and thus, subject to unwanted contamination. Second, thesample is exposed to air, which prevents the use of air-sensitivesamples. Third, there is a large amount of variability within a singleEM grid, between grids stained by a single user, and even morevariability between users.

The significance of the variability is so great that the preparation isfrequently compared to an art-form. Typically, this problem is overcomeby arbitrarily sampling regions of the grid until one can be found wherethe specimen subjectively looks the best. However, thisirreproducibility can lead to bias, staining artifacts, and poorsignal-to-noise, which can degrade image resolution and informationcontent.

A few examples of microfluidic systems have been published for samplepreparation of cryo-EM samples or for negative-stained samples (Jain etal., 2012; Kemmerling et al., 2012; Lu et al., 2009; Lu et al., 2014).However, these were highly specialized devices which would need to beredesigned for specific samples. Additionally, the designs relied onsample spraying techniques that can be disruptive to the structure ofmacromolecular complexes. Proteins are often prone to denaturation atthe air/water interface, and spraying techniques are limited to samplesthat are relatively insensitive to the interface, such as well-behavedsamples like ribosomes and GroEL. Ultimately, these protocols offered noquantitative assessment of the sample. Id.

Collectively the technologies mentioned above may have some merit toimprove throughput and reproducibility of sample preparation, but theyrequired the utilization of robotics thus hindering the methods'translation. Moreover, these advances are auxiliary additions to thesame preparation workflow.

Accordingly, what is needed is a device and method for preparing amicroscope sample that remove or minimize user variation and preventexposure of the sample to air. However, in view of the art considered asa whole at the time the present invention was made, it was not obviousto those of ordinary skill in the field of this invention how theshortcomings of the prior art could be overcome.

All referenced publications are incorporated herein by reference intheir entirety. Furthermore, where a definition or use of a term in areference, which is incorporated by reference herein, is inconsistent orcontrary to the definition of that term provided herein, the definitionof that term provided herein applies and the definition of that term inthe reference does not apply.

While certain aspects of conventional technologies have been discussedto facilitate disclosure of the invention, Applicants in no way disclaimthese technical aspects, and it is contemplated that the claimedinvention may encompass one or more of the conventional technicalaspects discussed herein.

The present invention may address one or more of the problems anddeficiencies of the prior art discussed above. However, it iscontemplated that the invention may prove useful in addressing otherproblems and deficiencies in a number of technical areas. Therefore, theclaimed invention should not necessarily be construed as limited toaddressing any of the particular problems or deficiencies discussedherein.

In this specification, where a document, act or item of knowledge isreferred to or discussed, this reference or discussion is not anadmission that the document, act or item of knowledge or any combinationthereof was at the priority date, publicly available, known to thepublic, part of common general knowledge, or otherwise constitutes priorart under the applicable statutory provisions; or is known to berelevant to an attempt to solve any problem with which thisspecification is concerned.

BRIEF SUMMARY OF THE INVENTION

The long-standing but heretofore unfulfilled need for a device andmethod for preparing an EM sample that remove or minimize user variationand prevent exposure of the sample to air is now met by a new, useful,and nonobvious invention.

The present disclosure is directed to a microfluidic sample preparationdevice, preferably for electron microscopy. Various embodiments mayallow for sealing of an EM grid, facile and reproducible delivery ofsample, followed by delivery of subsequent solutions that may benegative stains or other biological samples. According to variousembodiments, the EM grid may be contained in a grid chamber using aplurality of support barriers and may be gently and easily removed withan extraction divot disposed at least partially below the grid chamber.The fluid may be directed to the grid using channels integrated into theplatforms of the microfluidic system. Single or multiple grids may behoused in a platform, which may allow for high throughput testing. Forexample, a device with nine grids may require less than 1 μL of sampleper grid. This may allow more screening in circumstances where samplequantity is limited.

Various embodiments comprise a device to deliver air-sensitive samplesto an EM grid via an air tight chamber. This technology fills a nichefor which no similar technology currently exists. Traditionally, EMstaining is a tedious and time consuming task that offers littlereproducibility. The conventional staining method is done in a manualfashion in an open environment, which may introduce contamination, isnot viable for air-sensitive samples, and may be plagued by user-to-uservariations.

Various embodiments may comprise two platforms, which may be comprisedof etched pieces of glass, aligned to one another forming an internalchamber sized to house the EM grid. Integrated microfluidic channelsallow the sample to be delivered to the grid in an automated fashion.Timing may be introduced using automated or integrated valves allowingtime-dependent snapshots of the sample.

The long-standing but heretofore unfulfilled need for an EM samplepreparation device capable of sealing of an EM grid from exposure toair, facile and reproducible delivery of sample, followed by delivery ofsubsequent solutions that may be negative stains or other biologicalsamples, and methods for its use, are now met by a new, useful, andnonobvious invention.

These and other important objects, advantages, and features of theinvention will become clear as this disclosure proceeds.

The invention accordingly comprises the features of construction,combination of elements, and arrangement of parts that will beexemplified in the disclosure set forth hereinafter and the scope of theinvention will be indicated in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 depicts the conventional method of preparing a sample for EMimaging highlighting the degree of manual interaction required tocomplete the preparation.

FIG. 2 is an overhead view of an embodiment of a bottom platform.

FIG. 3 is an overhead view of the embodiment provided in FIG. 2 with theaddition of a microscope grid.

FIG. 4 is an embodiment of the novel method of preparing an EM sample.

FIG. 5A is a section view of section line A-A in FIG. 2 with theinclusion of the top platform.

FIG. 5B is a section view of section line A-A in FIG. 3 with theinclusion of the top platform.

FIG. 6A depicts an example of functionalized top and bottom platformsusing fluoropolymer.

FIG. 6B depicts an example of functionalized top and bottom platformsusing alkane chains.

FIG. 6C depicts an example of functionalized top and bottom platformsusing biotin-streptavidin reaction.

FIG. 7 is an exemplary embodiment of a mechanical clamp for securing thetop and bottom platforms together.

FIG. 8 is an embodiment of a bottom platform comprising multiple gridchambers.

FIG. 9 provides several embodiments of a bottom platform having multiplegrid chambers (top two rows) and fluidic timers (bottom row).

FIG. 10 is an exemplary embodiment of bottom platform illustrating thepossibility of combining multiple sets of grid chambers with each setcoupled to a microfluidic timer.

FIG. 11 is an exemplary embodiment of bottom platform illustrating theuse of a gradient generator with multiple grid chambers.

FIG. 12 is an exemplary embodiment of bottom platform illustrating theuse of a gradient generator with multiple grid chambers and anadditional sample inlet.

FIG. 13 depicts the use of an embodiment of a bottom platform with aseparate gradient generator platform.

FIG. 14 depicts an exemplary method for manufacturing the platforms. Thephotomasks on the left half are used to fabricate the platforms shown onthe right using photo lithography. In order to obtain two differentdepths, separate exposure and etching steps are implemented. First anextraction divot is etched into the glass and subsequently the gridchambers and channels are added. This may allow for the integration ofplumbing and an extraction pathway.

FIG. 15 illustrates low magnification (130×) images of artifactsresulting from conventional manual preparation (top row) and frompreparation according to the present invention (bottom row).

FIG. 16 provides a comparison between the samples prepared by hand (rowA) and the samples prepared by the present invention (row B) by way ofhigh magnification images.

FIG. 17 illustrates two-dimensional reference free class average ofKvβ2.1 particles picked from micrographs prepared using the samplepreparation device of the present disclosure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a partthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

The present invention includes a novel device and method for preparing asample for EM imaging. The present invention provides an innovativeapproach to 1) create a robust and reproducible method for negativestaining of EM grids; 2) automate preparation of multiple samplessimultaneously; and 3) integrate quantitative assessments of samplestability. The technologies described herein are suitable forapplications across the field of EM and will have a significant impacton a multitude of other biological systems.

As shown in FIG. 2, an embodiment of the novel apparatus includes bottomplatform 102 having inlet 104, outlet 106, and grid chamber 108 disposedbetween inlet 104 and outlet 106. Grid chamber 108 is in fluidcommunication with inlet 104 and outlet 106 through inlet channel 110and outlet channel 112, respectively. Bottom platform 102 furtherincludes a plurality of grid support barriers 114 disposed in gridchamber 108 and extraction divot 116 located at least partially withinthe outer perimeter of grid chamber 108. Bottom platform 102 alsopreferably includes at least one alignment marker 118.

Bottom platform 102 is preferably flat and may be comprised of generallyany material including, but not limited to glass, metals, ceramics,plastics, silicon, and elastomers. Fabrication may be achieved throughknown processes for creating microfluidic devices including, but notlimited to lithography, 3D printing, hot embossing, and milling. Thefabrication method is at least partially dependent on the structuralfeatures of the bottom platform and the material included in the bottomplatform.

As depicted in FIG. 3, EM grid 120 is intended to rest in grid chamber108 within the perimeter created by support barriers 114. The exemplaryembodiment shows grid chamber 108 as hexagonal in shape, but any shapeis contemplated. Support barriers 114 are strategically arranged tocreate a perimeter of a proper size for receiving and securing EM grid120 within grid chamber 108. The support barriers 114 minimize gridmovement while a pressure-driven fluid flow passes through grid chamber108. The fragile nature of EM grid 120 and its carbon coating requirethat EM grid 120 remain generally static as the pressurized flowovertakes EM grid 120. It was discovered that the absence of supportbarriers 114 resulted in EM grid 120 sliding around grid chamber 108upon application of fluid, thus causing the carbon film to tear.

In the depicted embodiment, support barriers 114 are arranged in agenerally circular fashion. The overall pattern/arrangement of barriers114 is dependent on the shape of EM grid 120, and thus, may be arrangedin a different pattern to secure EM grid 120 within grid chamber 108.

In an embodiment, the arrangement of barriers 114 must include a gapbetween barriers 114 sufficiently sized to account for extraction divot116 disposed within the gap. Extraction divot 116 allows for easyremoval of the fragile EM grid 120 using sharp-tipped forceps or asimilarly designed device. Extraction divot 116 further aids inpreventing EM grid 120 from sticking to bottom platform 102 when a userattempts to remove EM grid 120 from grid chamber 108.

Referring now to FIG. 4, bottom platform 102 is designed to receive topplatform 122. In its simplest form, top platform 122 is a simple barriersecured in overlying relation to bottom platform 102. Top platform 122includes inlet aperture 124 and outlet aperture 126 to provide a fluidpassage to the inlet 104 and outlet 106, respectively. Upon loading EMgrid 120 into chamber 108, all the externally conducted steps shown inFIG. 1 can be performed in a temporarily sealed environment minimizingthe exposure and the variability resulting from manual EM grid handling.EM grid 120 is preferably sealed into chamber 108 using bottom platform102 and top platform 122 to ensure accurate and reproducible flowpatterns for sample preparation

An embodiment of the novel method of EM sample preparation, using anembodiment of the novel device, is illustrated in FIG. 4. The sample isprepared according to the five general steps executed from left toright. In the first step, EM grid 120 is deposited within supportbarriers 114 located in grid chamber 108 in bottom platform 102. Topplatform 122 is secured in overlying relation to bottom platform 102with inlet and outlet apertures 124, 126 respectively aligned with inlet104 and outlet 106. In the second step, sample 103 is inserted into thesystem through inlet 124 using a fluid application device, such aspipette 128. The third step includes inserting stain 105 into the systemusing a fluid application device, such as pipette 128. The fourth stepcomprises drying the system using a gas inserted into aperture 124. OnceEM grid 120 is adequately dried, top platform 122 is removed. EM grid120 is removed from bottom platform 102 in the final step. Through theuse of the microfluidic platforms, all the preparation steps areintegrated into a single system overcoming the irreproducibilityobstacles prevalent in EM sample preparation.

In experimental testing, EM grid 120 would often stick to the topplatform 122 when top platform 122 was removed in step five above. Thefragility of the EM grid 120 became an issue when attempting to removeEM grid 120 from top platform 122. As a result, a preferred embodimentof the present invention, as depicted in FIGS. 5A-5B, includes topplatform 122 having etching in its lower surface that is similar to theetching in the upper surface of bottom plate 102. FIG. 5 provide asectional view along section line A-A shown in FIGS. 2-3 and illustrateshow fluid, depicted by arrows 130, passes into the system through inletaperture 124 and then out of the system through outlet aperture 126. Thefigures depict inlet and outlet channels 110, 112 as having a depthgenerally equal to the depth of grid chamber 108, but the channels canbe any size relative to the size of the grid chamber.

In an embodiment, the top platform is simply an inverted bottom platformto reduce manufacturing efforts. In yet another embodiment, top platform122 simply includes an extraction divot disposed in the lower surface toallow a user to remove an EM grid stuck to the top platform using anextraction tool.

To ensure proper alignment of the top and bottom platforms 122, 102, anembodiment of the present invention may include each platform having analignment marker 118 (See e.g. FIGS. 8-11 and 14). In an embodiment, thealignment marker on the top platform is designed to interact with thealignment marker on the bottom platform to prevent respective movementof the two platforms in the transversal and longitudinal directions. Thealignment markers are disposed at least on the upper surface of thebottom platform and on the lower surface of the top platform.

As is discussed below in the experiment section, there was a substantialbenefit from sealing the top and bottom platforms. Therefore, apreferred embodiment includes a sealed system to ensure accurate andreproducible flow patterns and also to protect the sample from theair-water interface. At the end of the sample preparation, however, theEM grid must be removed for visualization by EM. Therefore, a reversiblesealing method for the system is required.

The system may employ functionalized materials for sealing the system.The different substrates can be functionalized in a panel of ways toprovide different sample solvent/matrix compatibility. For example,functionalization methods include, but are not limited to fluorinefunctionalized surfaces (FIG. 6A), alkane functionalized surfaces (FIG.6B), biotin-streptavidin (FIG. 6C), carbon coating by sputtering, plasmaoxidized, and carbon coated and plasma oxidized. It should be noted thatthe plasma oxidation rendered the surface hydrophilic and led to rapidtransfer of solution between slides. This strategy can be used toaccelerate and guide the passage of fluid through the channels.

Alternatively, the bottom and top platforms may be temporarily sealedusing mechanical devices, including, but not limited, to a binder clip,clamps, manifolds that have a built in screw/clamping system, magnets,or a device integrated into the top and bottom platforms. In anembodiment, a gasket is disposed between the sandwiched top and bottomplatforms to seal the grid chamber and channels.

A particular example of a mechanical clamping device is provided in FIG.7. The device includes base platform 202 having recess 204 in whichbottom and top platforms 102,122 (not shown) are intended to rest.Clamping arms 206 force platforms 102, 122 together when in the securingconfiguration shown. Clamping arms 206 may be opened by rotating the topassembly arm 208 in an outboard direction, which removes the clampingforce on the platforms 102, 122. Bottom and top platforms 102,122 canthen be easily removed from recess 204.

Fluid (sample, stain, rinse, etc.) delivery and control can take onvarious forms depending on the type of sample being prepared and thegoals of the preparation. The method shown in FIG. 4 relies on pipette128 for the delivery of the sample and stain to the grid chamber 108.Other manual fluid delivery options include, but are not limited to, asyringe pump, a surface tension/capillary action driven fluid, a vacuumat the outlet aperture 126, peristatic pumps, and tilting the system torely on gravity to drive the fluid from inlet aperture 124 to outletaperture 126. Many of these pressure-driven systems would be implemented“off-chip,” but some could also be implemented directly within themicrofluidic system using micro-fabrication techniques. Some of thesemicro-fabrication techniques require multiple layers of the device,which is compatible with this system.

Fluid delivery may, alternatively, be automated. These automated measureinclude, but are not limited to, “on-chip” valves or pumps,electroosmotic flow—voltage driven, and the use of a pre-filled tubehaving pre-measured segments of the sample, stain, rinse, etc.

In an embodiment, the fluid delivery mechanisms are coupled to the inletaperture, and preferably also the outlet aperture, to maintain a sealedenvironment and ensure direct application into the system. The couplingcan be achieved according to any method known by a person havingordinary skill in the art for securing a fluid delivery system to amicrofluidic platform, including, but not limited to, press fittingtubes into the apertures, securing nano-port fitting in the apertures,and bonding a threaded reservoir into the apertures and then interfacingthe reservoir with tubing and fittings.

The removal of manual handling and manual application of fluids duringEM sample preparation, which is now possible with the present invention,opens the field of EM sample preparation to both high throughputproduction, microfluidic timers, and microfluidic gradient generators.It should be noted that the microfluidic features can be on the samedevice or a separate platform. A separate device having the microfluidicfeatures is may be desirable for easily interchanging the preparationplatforms.

Referring now to FIG. 8, an embodiment of the present invention providesa high throughput system for simultaneous preparation of multiple EMsamples and multi-screening from the same sample. Bottom platform 102includes twelve separate grid chambers 108 each with their own inlet 104and outlet 106. Obviously alignment of bottom platform 102 and topplatform (not shown) is critical when several grid chambers 108 aredisposed in each platform. Thus, each platform includes alignment marker118. Preferably alignment marker 118 on bottom platform 102 is designedto interconnect with an alignment marker on the top platform to preventlateral and longitudinal movement between the two platforms.

Referring now to FIG. 9, an embodiment may include multiple gridchambers in fluidic communication and arranged in series or in parallelto create microfluidic timers for fluid delivery/fluid interactionand/or to increase the production rate of EM samples. The first two rowsof bottom platforms 102 provides various examples of how multiplefluidly coupled grid chambers might be arranged. The third row of bottomplatforms 102 depicts exemplary embodiment of microfluidic timers usedwith a single grid chamber 108. As illustrated in the first two rows,each set of fluidly coupled grid chambers includes a single inlet 104and a single outlet 106. The grid chambers 108 may be arranged in anyconfiguration and may be coupled through various intermediate channels132. Furthermore, each grid chamber 108 preferably includes a pluralityof support barriers 114 for securing an EM grid within a particular gridchamber 108. Likewise, each grid chamber 108 preferably includes anextraction divot 116 for removing an EM grid from a particular gridchamber 108. These fluidly coupled grids significantly improve the speedat which samples can be prepared compared to the conventional manualpreparation.

The third row of bottom platforms 102 depicts exemplary embodiments ofmicrofluidic timers used with a single grid chamber. Two inlets 104 maybe used to create timed reactions, which occur while the injected fluidspass through inlet channel 110 to grid chamber 108. By using differentlength inlet/mixing channels 110, the time for the sample to reach gridchambers 108 and for the reaction to occur can vary.

Referring to FIG. 10, an embodiment of platform 102 may include gridchambers 108 in series with each series coupled to a microfluidic timercomprising two inlets 104 converging to inlet channel 110. Thisarrangement produces different incubation and mixing times and allowsfor complexes and reactions to reach different states before enteringeach sequential grid chamber. The time points of reaction can berecorded in a “snap-shot” by sequentially applying the sample to theseries of grids to capture time-dependent processes. Likewise, fixativecould be delivered at specific time increments to trap complexes atdifferent stages of activity.

An embodiment, as shown in FIG. 11, may employ a gradient generator tomix a panel of different conditions to study their effect on a structureof a complex/molecule. This can also be used to study time-dependentassembly mechanisms and reactions. A multitude of samples can be createdby adjusting the variable parameters, which include, but are not limitedto, the number of inputs and complex gradients, the mixing times andreaction times, the flow rates, and the amount of grids that can bescreened.

The embodiment of bottom platform 102 provided in FIG. 11 includes agradient between inlets 104 and grid chambers 108. As shown, fluid A isinserted into the left inlet and fluid B is inserted into the rightinlet. The gradient creates varying concentrations of the two fluids ineach grid chamber 108. The leftmost grid chamber has a low concentrationof fluid B and a high concentration of fluid A. The reverse is true atthe rightmost grid chamber and varying concentrations can be found inthe grid chambers between the two outer grid chambers.

An embodiment shown in FIG. 12 includes an additional stain deliveryinlet 134 to apply stain directly to the grid instead of flowing itthrough the gradient channels. The embodiment may also include softlithography valving (V1, V2, VS1-VS12), which are used to shunt the flowonly for the desired paths. This prevents backflow of stain for thesample delivery and allows the devices to be reused.

In an embodiment as shown in FIG. 13, bottom platform 102 may beseparate from gradient platform 140. Fluids are first inserted intogradient inlets 144, and after mixture, the fluid exists gradientoutlets 146. Outlets 146 are fluidly coupled (not depicted) to inlets104 for each grid chamber 108 in bottom platform 102. A separategradient platform allows for simplistic and efficient replication ofuse.

Experimental Research

The reagents used in the experiment included nitric acid, hydrogenperoxide, hydrofluoric acid, sodium hydroxide, ethanol, and(tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane. Ultrapuredeionized water was used for all solutions and sample preparation, andKvBeta was recombinantly expressed in and purified using known methods.

Fabrication. In order to get multiple depth steps in a single platform,the microfabrication steps were repeated twice on the same wafer (FIG.14). First, borofloat photoresist wafers with a layer of AZ1500 positivephotoresist on a chrome layer were exposed to 18 mW cm⁻¹ collimated UVradiation for 15 seconds through mylar, patterned photomask 150containing extraction divot 116. The exposed photoresist was removedwith AZ 400K Developer, diluted 1:3 in H₂O. The bottom chrome layer wasthen developed with a chrome etchant solution. The exposed glass wasthen etched in a 5:1:3 (v:v:v) mixture of H2O:HNO3:HF to a 40 μm depth.For the second step, the wafer was aligned with photomask 152 containingthe design with the channels 110, 112 and grid chamber 108. The featureswere developed and etched again to a depth of 110 μm. This produced achannel depth of 70 μm with an extraction divot of 40 μm. The totalchamber volume was 3 μL.

The same process was repeated for another chip, developing the mirrorreflection of the design features, this would serve as the topcomplement platform. All dimensions of the channels were verified usinga P-15 stylus profilometer. Fluid access holes were drilled with a 1.1mm diamond-tipped drill bit, after which the remaining photoresist andchrome were removed. The finished top platform was then fitted with ananoport that was attached using epoxy.

For the surface modification, the glass was cleaned by submerging in 5 MNaOH for 10 minutes. The surface was rinsed with water and dried withN₂. Subsequently the platforms were oxidized in a plasma cleaner for 2minutes. Immediately after, the platforms were placed in a vacuumdesiccator and (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilanewas deposited using a known method. Subsequently, the platforms wererinsed with water, dried with N₂, and stored in clean petri dishes atroom temperature until use.

Sample Preparation. The bottom glass platform was placed into thealuminum manifold. A carbon coated, copper grid was rendered hydrophilicusing a plasma cleaner and gently placed into the device chamber.Several 20 μL drops of buffer (20 mM Tris pH 8.0, 150 mM KCl, 1 mM2-mercaptoethanol) were distributed around the non-etched parts of theglass platforms. The top platform was aligned using the manifold andlowered on top of the bottom platform, in process displacing the bufferand creating a thin sealing layer. The top of the manifold was attachedand screwed down to seal the device. 20 μL of sample was loaded in theinlet and a vacuum was applied at the outlet to fill the chamber withsample. Alternatively, the sample could be delivered to the inlet usinga syringe with appropriate fitting. The vacuum was removed and thesample was left in the chamber for 10 seconds after which 50 μL ofuranium acetate stain was loaded into the inlet and carried through withvacuum. After 10 seconds, compressed air was blown into the inlet andused to dry the grid. The air also purged the thin film of bufferbetween the platforms, enabling the device to be opened and the gridextracted via the divot with a pair of forceps.

Electron Microscopy and Reconstruction. EM micrographs of Kvβ2.1 werecollected on a CM-120 BioTwin operating at 120 keV at room temperaturewith a nominal pixel size of 2.88 angstroms per pixel equipped with aTem-Cam F224 slow scan CCD camera. EM micrographs were uploaded to theAppion processing suite. Kvβ2.1 particles were picked in asemi-automatic fashion using the template picker FindEM. Two dimensional(2D) class averages were generated using the maximum likelihoodalignment algorithm within the Xmipp package.

Results and Discussion. Reproducibility in the staining process wasattained by integrating all the sample preparation steps into a singledevice that housed the EM grid (FIG. 4). Further advantages realizedthrough various embodiments include, but are not limited to: (1)simplicity of use and no requirement for further accessories, (2) easilyfabricated and reusable and (3) the system is amenable for integrationof further “on-chip” valving and plumbing.

After grid 120 was placed in the chamber, it was confined by supportbarriers 114 to prevent sliding and flow induced friction that couldtear the carbon film containing the sample. In absence of supportbarriers, EM grid 120 became torn upon application of the sample. Thesupport barriers preserved the grid integrity completely. Extractiondivot 116 also permitted easy grid access and extraction. Upondisassembling of the device, grid 120 would occasionally stick to topplatform 122. To address this problem, a divot 116 and set of barriers114 were etched into top platform 122 as well, permitting extractionwithout sticking.

In order to interface microfluidics with EM preparation, reversiblesealing of the grid inside a device is required. This was achieved bysilanizing the glass surface with(tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane, producing aneight carbon long, fluorinated moieties on the surface. This renderedthe glass both fluorophilic and hydrophobic. The fluorophilic surfacesof the two glass platforms interact with each other and form anon-covalent interaction, sufficient to seal the chip reservoir andchannels. The fluorophilic surfaces may prevent the sample from wickingin between the platforms, yet the force is sufficient to incorporatesyringe pump integration. The platforms appeared to seal better when afilm of buffer was introduced between the platforms. This is believed tobe due to an alignment of the fluorinated chains that might be in acollapsed state when dehydrated. In some embodiments, longer alkanechains with higher amount of fluorination may be used to strengthen theglass bond while maintaining reversible sealing.

Besides the improvement in the reproducibility of the staining, thecleanliness of the grids was improved and found to be free ofparticulate contamination. When making grids, contamination known as“crud” is the norm yet it consumes functional space on the grid and hasthe potential to interfere with the sample application and staining. Byincorporating all the steps into a single device as shown in FIG. 4,contamination was effectively eliminated.

The contamination of the grids was compared between the hand preparedgrids and the grids prepared using the device of the present invention,which is depicted in FIG. 15. All images were taken from separatelyprepared grids, however, the grids were prepared using the same sampleand on the same day. The second row of images corresponds to the gridsprepared using the present invention. As is shown, the grids arepredominantly clean, only infrequent particulates are observed, and thestain thickness is even throughout the grid. The top row of imagescorresponds to grids prepared by hand under the prior art methodology.When prepared by hand, abundant contamination with particulates anddebris are evident. In addition, vast differences between hand preparedgrids, including striations of different stain thicknesses producedduring the drying process, illustrate the variability in the stainingprocess. In contrast, the grids, produced using the device of thepresent disclosure, were reproducibly prepared, with similar stainthickness and in absence of contamination. Clearly the present inventionaids in sample preparation and will help transition this technology tonon-expert users.

The collective quality of the images acquired following preparation inthe device of the present invention was equal if not better to thoseprepared by hand. A magnification series comparing both preparations isshown in FIG. 16. The sample contamination is evident in the lowmagnification image prepared by hand (first image in row A).Nonetheless, stained KvBeta particles can be discerned at the highermagnifications. When using the device of the present invention, depictedin row B, the particles appear to be more evenly distributed with adenser stain. As the magnification increases, the Kvβ2.1 particles areseen as monodisperse and evenly stained yielding strong signal to noise.The cleaner surfaces provided with the device may increase the availablearea for imaging, minimize variability, and increase reproducibility ofthe staining methods. The final stain images at 52 k magnification arecomparable, with slightly improved stain coverage when using the device.

To illustrate the viability of this approach for structural biology, a2D class average of the Kvβ2.1 complex was performed (FIG. 17). Thereconstruction reveals structural motifs comparable to methods performedby hand and validates the coupling of microfluidics with EM samplepreparation.

Glossary of Claim Terms

Fluid Delivery Mechanism: is a device configured to transfer fluid fromone location to another location.

Gradient Generator: is a plurality of fluidic channels designed toincrease or decrease the concentration of a fluid observed in passingfrom one gradient outlet to another gradient outlet.

Microfluidic Timer Channel: is a fluidic channel having an indirectextended route between an inlet and the grid chamber.

Platform: is a generally rigid material, such as glass.

Stain: is a fluid used to artificially highlight tissue, microorganisms,and other biological structures for viewing, typically under amicroscope.

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Where a definition or use of a term in a reference, which isincorporated by reference herein, is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply.

The advantages set forth above, and those made apparent from theforegoing description, are efficiently attained. Since certain changesmay be made in the above construction without departing from the scopeof the invention, it is intended that all matters contained in theforegoing description or shown in the accompanying drawings shall beinterpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention that, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. A microscope grid preparation device, comprising:a bottom platform having upper and lower surfaces, the bottom platformfurther including: a grid chamber extending downwardly from the uppersurface; an extraction divot having at least a portion of the divotlocated within the grid chamber, a bottom surface residing at a depth inthe bottom platform that is greater than a depth of the grid chamber,and a width less than a width of the grid chamber, thereby enabling aportion of an extraction tool to pass under the grid chamber for easyremoval of a microscope grid within the grid chamber; a top platformhaving upper and lower surfaces, wherein the lower surface is configuredto rest in overlying relation to the bottom platform; a fluid inlet influid communication with the grid chamber; and a fluid outlet in fluidcommunication with the grid chamber.
 2. The device of claim 1, furthercomprising a plurality of support barriers disposed within the gridchamber and extending upwards from a base of the grid chamber towardsthe upper surface of the bottom platform, the plurality of supportbarriers arranged in a shape conducive for securing a microscope gridwithin the grid chamber.
 3. The device of claim 2, wherein the supportbarriers are arranged in a semi-circular formation.
 4. The device ofclaim 1, further comprising the top platform further having: an inletaperture creating a through hole between the upper surface and lowersurface of the bottom platform, thereby allowing fluid to pass from theupper surface of the top platform into the fluid inlet; and an outletaperture creating a through hole between the upper surface and lowersurface of the bottom platform, thereby allowing fluid to pass from thefluid outlet to the outlet aperture in the top platform.
 5. The deviceof claim 4, wherein the inlet and outlet apertures in the top platformare coupled to a fluid delivery mechanism.
 6. The device of claim 1,wherein the top and bottom platforms are configured to becometemporarily secured together to seal off the grid chamber from theenvironment.
 7. The device of claim 1, wherein the top platform includesan alignment marker on the lower surface, the bottom platform includesan alignment marker on the upper surface, and the alignment markers aredesigned to interact with each other when the upper surface of thebottom platform and the lower surface of the top platform are forcedtogether.
 8. The device of claim 1, wherein the top platform furtherincludes: a grid chamber extending upwardly from the lower surface; aplurality of support barriers disposed within the grid chamber andextending downwards from a base of the grid chamber towards the lowersurface of the top platform, the plurality of support barriers arrangedin a shape conducive for securing the microscope grid within the gridchamber; and an extraction divot having at least a portion of the divotlocated within the grid chamber, a top surface located closer towardsthe upper surface of the bottom platform than the base of the gridchamber, and a width less than a width of the grid chamber, therebyenabling a portion of the extraction tool to pass above the grid chamberfor easy removal of the microscope grid within the grid chamber.
 9. Thedevice of claim 1, further comprising the bottom platform having: asecond grid chamber extending downwardly from the upper surface; asecond plurality of support barriers disposed within the second gridchamber, wherein each support barrier extends upwards from a base of thesecond grid chamber towards the upper surface of the bottom platform andthe second plurality of support barriers are arranged in a shapeconducive for securing a microscope grid within the second grid chamber;a second extraction divot having at least a portion of the second divotlocated within the second grid chamber, a bottom surface residing at adepth in the bottom platform that is greater than a depth of the secondgrid chamber, and a width less than a width of the second grid chamber,thereby enabling a portion of an extraction tool to pass under thesecond grid chamber for easy removal of the microscope grid disposedwithin the grid chamber; an inlet channel in fluid communication withthe second grid chamber; and an outlet channel in fluid communicationwith the second grid chamber.
 10. The device of claim 9, furthercomprising a gradient generator disposed within the top surface of thebottom platform, the gradient generator having two or more gradientinlets for receiving fluids and two or more gradient outlets with eachgradient outlet in fluid communication with one inlet.
 11. The device ofclaim 1, wherein the bottom plate further includes a second inlet influid communication with the grid chamber and the inlet through amicrofluidic timer channel.
 12. A method for preparing microscopesamples, comprising: inserting a microscope grid within a grid chamberin a bottom platform; temporarily securing a top platform overtop thebottom platform; delivering a first fluid into an inlet aperture,wherein the inlet aperture is in fluid communication with grid chamber;propelling the first fluid through the grid chamber and out through anoutlet aperture in fluid communication with the grid chamber; anddelivering a second fluid into the inlet aperture and propelling thesecond fluid through the grid chamber and out through the outletaperture.
 13. The method of claim 12, further comprising the step ofdrying the microscope grid through insertion of a gas into the inlet.14. The method of claim 12, further comprising the steps of removing thetop platform and removing the microscope grid from the grid chamber. 15.The method of claim 14, wherein the step of removing the microscope gridincludes inserting a distal end of an extraction tool into an extractiondivot to aid in grasping the microscope grid, wherein the extractiondivot extends deeper into the bottom platform than the grid chamber andincludes at least a portion disposed within the grid perimeter.
 16. Themethod of claim 12, wherein the first fluid includes a sample ofinterest.
 17. The method of claim 12, wherein the second fluid is astain.
 18. The method of claim 12, further comprising the step ofaligning a grid chamber in the top platform with the grid chamber in thebottom platform before temporarily securing the top platform to thebottom platform.
 19. The method of claim 12, wherein the step oftemporarily securing a top platform overtop the bottom platform includestemporarily sealing the top platform and bottom platform together.
 20. Amicroscope grid preparation device, comprising: a bottom platform havingupper and lower surfaces, the bottom platform further including: a gridchamber extending downwardly from the upper surface; a plurality ofsupport barriers disposed within the grid chamber and extending upwardsfrom a base of the grid chamber towards the upper surface of the bottomplatform, the plurality of support barriers arranged in a shapeconducive for securing a microscope grid within the grid chamber; a topplatform having upper and lower surfaces, wherein the lower surface isconfigured to rest in overlying relation to the bottom platform; a fluidinlet in fluid communication with the grid chamber; and a fluid outletin fluid communication with the grid chamber.